Antiviral Activity of Zinc Oxide Nanoparticles against SARS-CoV-2

The highly contagious SARS-CoV-2 virus is primarily transmitted through respiratory droplets, aerosols, and contaminated surfaces. In addition to antiviral drugs, the decontamination of surfaces and personal protective equipment (PPE) is crucial to mitigate the spread of infection. Conventional approaches, including ultraviolet radiation, vaporized hydrogen peroxide, heat and liquid chemicals, can damage materials or lack comprehensive, effective disinfection. Consequently, alternative material-compatible and sustainable methods, such as nanomaterial coatings, are needed. Therefore, the antiviral activity of two novel zinc-oxide nanoparticles (ZnO-NP) against SARS-CoV-2 was investigated in vitro. Each nanoparticle was produced by applying highly efficient “green” synthesis techniques, which are free of fossil derivatives and use nitrate, chlorate and sulfonate salts as starting materials and whey as chelating agents. The two “green” nanomaterials differ in size distribution, with ZnO-NP-45 consisting of particles ranging from 30 nm to 60 nm and ZnO-NP-76 from 60 nm to 92 nm. Human lung epithelial cells (Calu-3) were infected with SARS-CoV-2, pre-treated in suspensions with increasing ZnO-NP concentrations up to 20 mg/mL. Both “green” materials were compared to commercially available ZnO-NP as a reference. While all three materials were active against both virus variants at concentrations of 10–20 mg/mL, ZnO-NP-45 was found to be more active than ZnO-NP-76 and the reference material, resulting in the inactivation of the Delta and Omicron SARS-CoV-2 variants by a factor of more than 106. This effect could be due to its greater total reactive surface, as evidenced by transmission electron microscopy and dynamic light scattering. Higher variations in virus inactivation were found for the latter two nanomaterials, ZnO-NP-76 and ZnO-NP-ref, which putatively may be due to secondary infections upon incomplete inactivation inside infected cells caused by insufficient NP loading of the virions. Taken together, inactivation with 20 mg/mL ZnO-NP-45 seems to have the greatest effect on both SARS-CoV-2 variants tested. Prospective ZnO-NP applications include an antiviral coating of filters or PPE to enhance user protection.


Introduction
In 2019, a new infectious disease, COVID-19, caused by SARS-CoV-2, spread rapidly around the world, posing major challenges for healthcare systems, economy and society. The highly contagious pathogen is mainly transmitted via respiratory droplets and aerosols during direct person-to-person contact [1] and, due to its high stability, via contaminated surfaces [2]. Approaches to combat the pandemic comprise reducing exposure and the Compared to current knowledge on the antibacterial activity of ZnO-NPs [8], data on their antiviral activity are limited. Therefore, the aim of the present study was to investigate the antiviral activity of two ZnO-NPs, ZnO-NP-45 and ZnO-NP-76, against SARS-CoV-2 including relevant variants in cell culture-based virus neutralization assays. The nanoparticles tested were prepared using an environmentally friendly, highly efficient method. The synthesis uses salts (nitrates, chlorates and sulfates) as a starting material and whey as a chelating agent instead of conventional analytical reagents [31]. The results of this research may form the basis for the development of an antiviral coating for PPE and filters to improve protection and reduce the transmission of pathogens such as SARS-CoV-2.

Metabolic Interference Assay
To investigate the metabolic effect of ZnO-NPs on Calu-3 cells, we performed the resazurin reduction assay [32][33][34] to determine nanoparticle concentrations that do not lead to cytotoxic or metabolic effects. This is crucial since too high concentrations could inhibit cell function, which could also interfere with virus replication in the cells and could lead to false positive results in the neutralization assays. The assay was performed under the same experimental conditions as the virus neutralization assays but without SARS-CoV-2. In brief, the metabolic activity of Calu-3 cells was measured 48 h after treatment with suspensions of ZnO-NP-45 and ZnO-NP-76 at seven different concentrations (1 mg/mL-100 mg/mL) and was normalized to the metabolic activity of untreated cells (Figure 1). The workflow is illustrated in Figure S1 (Supplementary Files).
gate the antiviral activity of two ZnO-NPs, ZnO-NP-45 and ZnO-NP-76, against SARS-CoV-2 including relevant variants in cell culture-based virus neutralization assays. The nanoparticles tested were prepared using an environmentally friendly, highly efficient method. The synthesis uses salts (nitrates, chlorates and sulfates) as a starting material and whey as a chelating agent instead of conventional analytical reagents [31]. The results of this research may form the basis for the development of an antiviral coating for PPE and filters to improve protection and reduce the transmission of pathogens such as SARS-CoV-2.

Metabolic Interference Assay
To investigate the metabolic effect of ZnO-NPs on Calu-3 cells, we performed the resazurin reduction assay [32][33][34] to determine nanoparticle concentrations that do not lead to cytotoxic or metabolic effects. This is crucial since too high concentrations could inhibit cell function, which could also interfere with virus replication in the cells and could lead to false positive results in the neutralization assays. The assay was performed under the same experimental conditions as the virus neutralization assays but without SARS-CoV-2. In brief, the metabolic activity of Calu-3 cells was measured 48 h after treatment with suspensions of ZnO-NP-45 and ZnO-NP-76 at seven different concentrations (1 mg/mL-100 mg/mL) and was normalized to the metabolic activity of untreated cells (Figure 1). The workflow is illustrated in Figure S1 (Supplementary Files).
ZnO-NP-45 and ZnO-NP-76 had no major effect on metabolic activity and viability of the cells at concentrations of up to 20 mg/mL. Higher concentrations (40 mg/mL-100 mg/mL) led to reduced metabolic activity of the cells, but even at 100 mg/mL ZnO-NP-45 and ZnO-NP-76, the cells still showed approximately 30-35% of metabolic activity, compared to untreated cells.
Based on these results, 20 mg/mL, 10 mg/mL and 5 mg/mL ZnO-NP-45 and ZnO-NP-76 were selected for the SARS-CoV-2 neutralization assays.  Calu-3 cells were seeded into 48-well plates 48 h before the assay. ZnO-NPs were dispersed in modified Eagle's medium (MEM) (without fetal calf serum (FCS)) and incubated for 1 h at 37 • C under continuous shaking at 300 min −1 . Subsequently, the suspensions were centrifuged, and the supernatant was applied to the cells for 1 h at 37 • C and 5% CO 2 . Then the cells were washed, fresh medium (+10% FCS) was added, and the metabolic activity was measured with a resazurin assay after 48 h incubation at 37 • C and 5% CO 2 . Columns represent the relative metabolic activity of the cells (%) normalized to the metabolic activity of control cells without treatment.
ZnO-NP-45 and ZnO-NP-76 had no major effect on metabolic activity and viability of the cells at concentrations of up to 20 mg/mL. Higher concentrations (40 mg/mL-100 mg/mL) led to reduced metabolic activity of the cells, but even at 100 mg/mL ZnO-NP-45 and ZnO-NP-76, the cells still showed approximately 30-35% of metabolic activity, compared to untreated cells.

Characterization of Zinc Oxide Nanoparticlets
ZnO-NPs were synthesized as described in Material and Methods and characterized using UV-VIS spectroscopy, scanning transmission electronic microscopy (STEM) and dynamic light scattering (DLS).
UV-VIS spectroscopy is often used to determine the size of nanoparticles. In the case of ZnO as a semiconductor, the initial and final absorbance wavelengths are closely related to the band gap of the particles, which is inversely proportional to their size. UV-VIS spectroscopy ( Figure 2) comparing the two nanomaterials presented a faster decay in absorbance with increasing wavelength in the case of ZnO-NP-45, indicating the presence of smaller nanoparticles in ZnO-NP-45, compared to ZnO-NP-76 ( Figure 3). seeded into 48-well plates 48 h before the assay. ZnO-NPs were dispersed in modified Eagle s medium (MEM) (without fetal calf serum (FCS)) and incubated for 1 h at 37 °C under continuous shaking at 300 min −1 . Subsequently, the suspensions were centrifuged, and the supernatant was applied to the cells for 1 h at 37 °C and 5% CO2. Then the cells were washed, fresh medium (+10% FCS) was added, and the metabolic activity was measured with a resazurin assay after 48 h incubation at 37 °C and 5% CO2. Columns represent the relative metabolic activity of the cells (%) normalized to the metabolic activity of control cells without treatment.

Characterization of Zinc Oxide Nanoparticlets
ZnO-NPs were synthesized as described in Material and Methods and characterized using UV-VIS spectroscopy, scanning transmission electronic microscopy (STEM) and dynamic light scattering (DLS).
UV-VIS spectroscopy is often used to determine the size of nanoparticles. In the case of ZnO as a semiconductor, the initial and final absorbance wavelengths are closely related to the band gap of the particles, which is inversely proportional to their size. UV-VIS spectroscopy ( Figure 2) comparing the two nanomaterials presented a faster decay in absorbance with increasing wavelength in the case of ZnO-NP-45, indicating the presence of smaller nanoparticles in ZnO-NP-45, compared to ZnO-NP-76 ( Figure 3). The hydrodynamic sizes and zeta potentials of the nanoparticles were determined using DLS and were measured with the ZetaSizer Nano ZS. Both ZnO-NP-45 and ZnO-NP-76 exhibited a positive zeta potential (28.87 ± 0.45 and 14.10 ± 0.20, respectively). In contrast, the reference particles exhibited a negative zeta potential (−1.69 ± 1.27) (Table 1). Although positively charged nanoparticles have been reported to be more cytotoxic than negatively charged particles [35], we did not observe cytotoxic effects at concentrations up to 20 mg/mL, as confirmed by the metabolic interference assay (Figure 1).
The size distribution of the nanoparticles was determined by DLS weighted for number and intensity ( Figure S3 (Table 1).
STEM images of ZnO-NP-45 displayed a polydisperse mix with a size distribution of 45 ± 15 nm ( Figure 3A  The hydrodynamic sizes and zeta potentials of the nanoparticles were determined using DLS and were measured with the ZetaSizer Nano ZS. Both ZnO-NP-45 and ZnO-NP-76 exhibited a positive zeta potential (28.87 ± 0.45 and 14.10 ± 0.20, respectively). In contrast, the reference particles exhibited a negative zeta potential (−1.69 ± 1.27) (Table 1). Although positively charged nanoparticles have been reported to be more cytotoxic than negatively charged particles [35], we did not observe cytotoxic effects at concentrations up to 20 mg/mL, as confirmed by the metabolic interference assay (Figure 1). Table 1. Hydrodynamic sizes and zeta potential of ZnO-NP-45, ZnO-NP-76 and ZnO-NP-ref. DLS: Average ± SD of mean values from intensity-and number-weighted distribution analyses of three measurements. Zeta potential: average ± SD in mV determined when suspended in Millipore water. PDI: Polydispersity index indicating the size homogeneity ranging from 0 (all particles are the same size) to 1 (highly polydisperse sample with wide range of particle sizes).

Sample
Size ( measurements. Zeta potential: average ± SD in mV determined when suspended in Millipore water. PDI: Polydispersity index indicating the size homogeneity ranging from 0 (all particles are the same size) to 1 (highly polydisperse sample with wide range of particle sizes).

Antiviral Activity of ZnO-NPs on SARS-CoV-2 Delta
The virus-neutralizing activities of the two different ZnO-NPs were analyzed in three independent assays, summarized in Figure 4a-d. The detailed experimental procedure is  (Table 1).
STEM images of ZnO-NP-45 displayed a polydisperse mix with a size distribution of 45 ± 15 nm ( Figure 3A,B). Images of the ZnO-NP-76 nanomaterial showed particles of about 76 ± 16 nm ( Figure 3C,D). Both ZnO-NPs were observed to form agglomerates in suspension. The reference nanomaterial ZnO-NP-ref were NPs of <100 nm and also tended to form agglomerates in suspension. All nanoparticles are shown with the maximum feasible magnification. The size differences of the nanoparticles between STEM and DLS are a result of their strong agglomeration. Energy dispersive X-ray analysis (EDX) intensity maps for the three NPs are shown in Figure S4 (Supplementary Files). Therefore, based on this analysis, NPs were free from trace metals such as Al, Ag or other heavy metals and offer biocompatibility and low cytotoxicity.

Antiviral Activity of ZnO-NPs on SARS-CoV-2 Delta
The virus-neutralizing activities of the two different ZnO-NPs were analyzed in three independent assays, summarized in Figure 4a-d. The detailed experimental procedure is illustrated in Figure S2 (Supplementary Files). For the neutralization assays, the NPs were pre-incubated with the virus for 1 h at 37 • C and subsequently centrifuged. Samples were taken from the supernatant of each tube to determine the virus input (VI) used for the infection of Calu-3 cells. The amount of virus RNA at 48 h after infection was determined via RT-qPCR in the cell culture supernatant, and virus copy numbers were calculated using an international reference standard. In addition, a commercial ZnO nanomaterial (Sigma-Aldrich; ZnO-NP-ref) was analyzed as a reference sample at concentrations of 20 mg/mL, 10 mg/mL and 5 mg/mL, respectively (Figure 4e,f). As a positive control for all assays, cells were infected with the same virus copy numbers as pre-incubated with the NPs but without NP pre-incubation. Non-infected cells were used as negative control.
Int. J. Mol. Sci. 2023, 23, x FOR PEER REVIEW 6 of 18 illustrated in Figure S2 (Supplementary Files). For the neutralization assays, the NPs were pre-incubated with the virus for 1 h at 37 °C and subsequently centrifuged. Samples were taken from the supernatant of each tube to determine the virus input (VI) used for the infection of Calu-3 cells. The amount of virus RNA at 48 h after infection was determined via RT-qPCR in the cell culture supernatant, and virus copy numbers were calculated using an international reference standard. In addition, a commercial ZnO nanomaterial (Sigma-Aldrich; ZnO-NP-ref) was analyzed as a reference sample at concentrations of 20 mg/mL, 10 mg/mL and 5 mg/mL, respectively (Figure 4e,f). As a positive control for all assays, cells were infected with the same virus copy numbers as pre-incubated with the NPs but without NP pre-incubation. Non-infected cells were used as negative control. All virus copy numbers are available in the Supplementary Files (Tables S1-S4). ZnO-NP-45 The pre-incubation of SARS-CoV-2 Delta with different concentrations of ZnO-NP-45 resulted in a concentration-dependent reduction in the number of viral copies in the supernatant after centrifugation (VI) as compared to the copy numbers in the untreated positive control (pos Ctrl) (Figure 4a). The dose-dependent reduction of the virus particles may be explained by the adsorption of the viruses to the NP surfaces and the loss of copies after centrifugation due to precipitation. Virus particles present in the supernatant were further tested for their infectivity by using the supernatant as virus input (VI) for infecting Calu-3 cells. At 48 h after infection, the virus concentration was measured by RT-qPCR in the cell culture supernatant to assess virus replication (Figure 4b). Virus concentrations in the cell culture supernatant of infected cells were reduced by more than 10 6 times after treatment with 20 mg/mL ZnO-NP-45 in all samples compared to cells infected with the virus without NP-pre-treatment (pos Ctrl). Treatment with 10 mg/mL ZnO-NP-45 also led to a reduction of replicating virus numbers, but not in all tested samples. At a concentration of 5 mg/mL ZnO-NP-45, a larger variation was observed in the individual experimental replicates, ranging from strong virus inactivation to high replication comparable to the positive control.

ZnO-NP-76
As already detected with ZnO-NP-45, pre-incubation of SARS-CoV-2 Delta with ZnO-NP-76 resulted in a similar dose-dependent reduction of virus copies in the VI after centrifugation, compared to the not pre-treated positive controls (Figure 4c). The effect seemed to be concentration-dependent since it was observed for all three assays and both NP preparations. Although the adsorption of the viral particles to ZnO-NP-45 and ZnO-NP-76 was comparable, they showed different neutralizing effects. Pre-treatment of SARS-CoV-2 Delta with 20 mg/mL ZnO-NP-76 showed similar virus-neutralizing activity to 10 mg/mL ZnO-NP-45, with a reduction of virus copies in many, but not all experimental replicates (Figure 4d

Immunohistochemistry
To demonstrate the virus-neutralizing effect of the NPs with a second independent read-out in addition to virus copy number calculations based on RT-qPCR, we immunohistochemically (IHC) stained the infected cells with an antibody against the SARS-CoV-2 nucleocapsid ( Figure 6). As expected, high numbers of infected cells were visible in all positive control (pos Ctrl) wells. Pre-treatment with ZnO-NP-45 at 20 and 10 mg/mL showed no infected cells, which is in line with the RT-qPCR results. In samples incubated with 5 mg/mL ZnO-NP-45, some wells showed infected cells. This outcome is in line with the virus copy numbers after the RT-qPCR (Figure 5b). For ZnO-NP-76 at 20 and 5 mg/mL, the IHC results confirmed data obtained by RT-qPCR showing both infected and non-infected cells in the stained wells. After treatment with 10 mg/mL ZnO-NP-76, no infected cells were detected. Furthermore, for the three concentrations of the reference nanoparticles, no infected cells were seen after the IHC staining which well reflects the very low virus copy numbers after the RT-qPCR (Figure 5f). All positive control wells were infected, while all negative control wells showed no infected cells.

Immunohistochemistry
To demonstrate the virus-neutralizing effect of the NPs with a second independent read-out in addition to virus copy number calculations based on RT-qPCR, we immunohistochemically (IHC) stained the infected cells with an antibody against the SARS-CoV-2 nucleocapsid ( Figure 6). As expected, high numbers of infected cells were visible in all positive control (pos Ctrl) wells. Pre-treatment with ZnO-NP-45 at 20 and 10 mg/mL showed no infected cells, which is in line with the RT-qPCR results. In samples incubated

Discussion
In this study, we examined the antiviral activity of two novel ZnO-NPs (ZnO-NP-45 and ZnO-NP-76) against the Delta and Omicron variants of SARS-CoV-2 in several independent cell-culture-based experimental series.
These nanomaterials are of great interest due to their physical and chemical properties, low cytotoxicity and sustainable manufacturing process, which allows an environmentally friendly "green" synthesis using salts (nitrates, chlorates and sulfates) as a starting material and whey as a chelating agent instead of conventional analytical reagents. Additionally, the manufacturing process allows doping of the ZnO with other antiviral materials, such as Ag or Cu, which could provide additional antiviral activities for future research and applications. In doped NPs, the atoms of the dopant are located inside the ZnO lattice and not on its surface. To our knowledge, such combinations have not yet been tested.
Our results show that ZnO-NP-45 inhibits replication of SARS-CoV-2 by a factor of up to 10 6 when incubated at a concentration of 20 mg/mL for 1 h prior to cell infection. This high factor of virus inactivation even exceeds the requirements for disinfectants since, according to the Robert Koch Institute (RKI) and the guideline of the German Association for the Control of Virus Diseases (DVV), a reduction of at least 10 4 is required for virus disinfection [36]. Lower concentrations of ZnO-NP-45 or ZnO-NP-76 also reduced virus replication, but not in all samples tested. In particular, after treatment with ZnO-NP-76, large differences in virus inactivation were observed at all concentrations tested, ranging from a strong reduction of infectious virus to high replication. This heterogeneity could be explained by incomplete virus inactivation after nanoparticle pre-treatment as well as by secondary infections occurring during the virus neutralization assay. This may be the case because within the 48 h of incubation of infected cells, even a few virus-replicating cells produce sufficient virus particles that may lead to secondary infections of most cells resulting in very high virus copy numbers as determined by RT-qPCR and also seen in IHC.
The reference nanoparticles ZnO-NP-ref also reduced the number of infectious virus particles but seemed to exhibit a stronger antiviral effect on the Omicron variant, as shown in Figure 5. Inactivation of infectious virus was observed for all concentrations and all replicates. With SARS-CoV-2 Delta, only a small number of replicates was inactivated at all NP concentrations. NP-ZnO-ref and NP-ZnO-76 are comparable in their inconsistent antiviral effect on the Delta variant, while 20 mg/mL ZnO-NP-45 resulted in an inactivation of infectious particles by a factor of 10 6 in all replicates. For the Omicron variant, a constant inactivation using ZnO-NP-45 was observed at 20 mg/mL and also at 10 mg/mL. Therefore, inactivation with 20 mg/mL ZnO-NP-45 seems to have the greatest effect on both SARS-CoV-2 variants tested.
We also noticed poor dispersion of the nanoparticles in the medium, leading to unstable suspensions, which made it difficult to standardize the experimental conditions, which may also contribute to some variations. To reduce this problem, each nanoparticle suspension was mixed immediately before use for each working step. During pre-treatment, the samples were shaken at 300 min −1 to keep as many particles as possible suspended and to minimize precipitation before centrifugation.
Pre-treatment of the virus with nanoparticles led to binding and co-precipitation of the virus and nanoparticles after centrifugation. This interaction was dose-dependent and reduced the VI used for the infection assay performed with the supernatant after pretreatment and centrifugation. Although the pre-treatment and centrifugation reduced virus copy numbers by a factor of up to 10 4 , the reduction of virus replication was up to a factor of 10 6 , which indicates that in addition to adsorption to its surface, the nanoparticles tested reduced virus infectivity by further inactivation. This appears to be a chemical antiviral effect on the virus that could inhibit infectivity when incubated together for one hour. The reference NPs (ZnO-NP-ref) also demonstrated an antiviral effect, as the reduction in viral replication cannot be explained only by particle loss in the VI after pre-incubation and centrifugation. Whether nanoparticles that were not precipitated by centrifugation exert some additional effect on the cells used for the neutralization assay cannot be excluded.
However, the metabolic interference assay performed excluded major interference with cell function at concentrations of up to 20 mg/mL nanoparticles.
The different antiviral effects of the two nanoparticle preparations might be explained by their differences in size distribution. As revealed by TEM and DLS, ZnO-NP-45 is composed of particles with sizes from approximately 20 nm to 80 nm, whereas ZnO-NP-76 is monodispersed and contains particles of about 80 nm. The enhanced virus binding and neutralization activity of ZnO-NP-45 might, therefore, be a consequence of the greater total reactive surface than that of ZnO-NP-76.
Previous studies on ZnO-NP have primarily focused on their inhibitory effect on various bacteria. For this reason, data on the effect of ZnO-NP on viruses are less common in the literature and are not yet available for SARS-CoV-2 variants. One possible antiviral effect could be hydrolyzation and inactivation of SARS-CoV-2 bound to ZnO-NPs by the alkaline zinc oxide. Another observation was made for the influenza virus H1N1. ZnO-NPs, both uncoated and PEGylated, were found to inhibit the intracellular replication of the H1N1 virus after cell infection by a mechanism possibly related to the release of Zn 2+ ions [27]. No comparable observations were made for SARS-CoV-2, but a similar mechanism can be expected and needs further research. The antiviral activity of ZnO-NPs on SARS-CoV-2 was already shown with VeroE6 cells, reporting severe damage to the viral envelope by free radicals causing oxidative stress to SARS-CoV-2 [29]. Reactive oxygen species (ROS) generated through photocatalysis can destroy proteins, lipids, carbohydrates and nucleic acids, ultimately leading to viral inactivation [37].
All of the mechanisms described here could be responsible for inactivating the virus, but there is no specific evidence of which chemical mechanism was responsible for the virus inactivation observed in our experiments.
In conclusion, the findings of this study clearly demonstrate a high antiviral activity of ZnO nanoparticles against the two SARS-CoV-2 variants Delta and Omicron, which is based on adsorption and an additional not yet defined antiviral effect. Particularly the ZnO-NP-45 nanomaterial, which is more active than the ZnO-NP-76, could be a promising material for future usage as a surface coating for antiviral PPE, especially coverall suits, face masks, and antiviral filters that could be used for air-conditioning and room ventilation systems.

Synthesis and Characterization of Zinc Oxide Nanoparticles
The two zinc oxide nanoparticle types, ZnO-NP-45 and ZnO-NP-76, were synthesized using a modified green sol-gel method, as described by Soares et al. in 2020 [31]. In brief, ZnO nanoparticles (NPs) were successfully synthesized by a whey-assisted sol-gel method. The composition of the gel and the subsequent calcination temperature showed efficiency in controlling the growth of nanocrystals. The sustainable method proved to be highly efficient for the synthesis of crystalline ZnO-NPs. As reference, commercially available ZnO-NPs <100 nm particle size (Cat# 544906, Sigma-Aldrich, Saint Louis, MO, USA) were used in separate neutralization assays.
The hydrodynamic sizes and zeta potentials of the ZnO-NPs were evaluated using dynamic light scattering (DLS). A concentration of 100 µg/mL of NPs was prepared in Mil-lipore water, and the measurements were performed using the ZetaSizerNano ZS (Malvern Panalytical, Malvern, UK) and the ZetaSizer Software (Malvern, 7.03, Malvern Panalytical, Malvern, UK), modulating the settings for refractive index of the NP composition and dispersant (2.0 for zinc oxide). The primary particle size and elemental composition of the ZnO-NPs were further determined by scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (XPS). For measurement, 2 µL of a 10 µg/mL NP dispersion was dried overnight on a lacy carbon-coated copper TEM grid and imaged using the JEM F200 (JEOL, Freising, Germany) electron microscope in STEM mode operated at 200 kV. Primary particle size was determined by calculating the mean ± SD of minimum 10 particles via image processing with the ImageJ software (NIH, Bethesda, MD, USA) and manual measuring. EDX intensity maps were acquired with a beam current of 0.1 nA and a beam diameter of 0.16 nm.

Dispersion of Zinc Oxide Nanoparticles
NPs were washed with 70% EtOH and air dried. The required amount of each NP preparation was weighed into 5 mL Eppendorf tubes and suspended in MEM medium, initially without FCS, to avoid unspecific adsorption of the NPs to BSA and other proteins present in the serum. The tubes were mixed for 30 s. using a vortex mixer to minimize agglomeration. It was observed that the nanoparticles precipitate within a short time. Changes in pH or suspension in other solvents did not improve the stability of the NP suspensions. The tubes were mixed before each working step to keep the NPs in suspension during the experiments. During pre-incubation of NPs and SARS-CoV-2, tubes were gently shaken at 300 min −1 .

Metabolic Interference Assay
The possible metabolic interference of ZnO-NP on Calu-3 (human lung epithelial cells) cells was evaluated using the resazurin reduction assay. Resazurin is a non-toxic dye that is reduced in metabolically active cells to fluorescent resorufin. Its concentration is measured fluorometrically at an excitation wavelength of 530 nm and an emission wavelength of 590 nm [32][33][34]. The optimal excitation wavelength for this assay in our experiments was found to be 485 nm. A stock solution of 1 mM resazurin (Sigma Aldrich R7017-1G, Merck KGaA, Darmstadt, Germany) was prepared in phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA), sterile filtered using 0.2 µm syringe filters (Thermo Fisher Scientific, Waltham, MA, USA) and stored at 4 • C protected from light. To determine the possible metabolic interference, the resazurin assay was performed following the same conditions as the neutralization assays but without the virus ( Figure S1, Supplementary Files). Calu-3 cells were seeded into 48-well microtiter plates (Corning Incorporated, Kennebunk, ME, USA) at a density of 30,000 cells per well and incubated at 37 • C and 5% CO 2 48 h before the assay. Suspensions of decreasing ZnO-NP concentrations (100, 80, 40, 20, 10, 5 and 1 mg/mL) were prepared in MEM medium containing 2% L-glutamine and 1% Penicillin-Streptomycin. ZnO-NP were incubated in medium for 1 h at 37 • C and 300 min −1 and subsequently centrifuged at 13,000× g for 5 min. The supernatant was collected and added to Calu-3 cells at 37 • C and 5% CO 2 for 1 h. Then, the cells were washed with MEM and fresh medium containing 10% FCS, 2% L-glutamine and 1% Penicillin-Streptomycin, was added. Cells without ZnO-NP treatment were used as references. The plate was incubated at 37 • C and 5% CO 2 for 48 h. To measure the metabolic activity of the cells, resazurin was added to each well at a final concentration of 10 µM. The fluorescent signal was recorded at a wavelength of 485/590 nm over a time period of 2 h at 37 • C using a microplate reader (BioTek Synergy 4, Szabo-Scandic HandelsgmbH, Vienna, Austria). The cell metabolic activity was measured 48 h after ZnO-NP treatment for each concentration. Linear regression equations of the recorded signals were generated, and the resulting slopes were normalized to the reference and plotted in a graph. in five replicates. Virus without NP pre-treatment was used as positive control, and non-infected cells served as negative control for the assay.
After infection, the cells were washed with MEM (no FCS), and fresh MEM (10% FCS) was added to the cells. After 48 h incubation at 37 • C and 5% CO 2 , supernatant of each well was collected to determine virus concentrations at t48 (timepoint 48 h after infection) via RNA isolation and RT-qPCR. A schematic workflow of the neutralization assay is shown in Figure S2 (Supplementary Files).

RNA Isolation and Reverse Transcription Quantitative PCR (RT-qPCR)
SARS-CoV-2 viral RNA from cell culture supernatant was extracted using the QIAamp ® Viral RNA Mini Kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer's recommendations. RNA samples were eluted with 40 µL Milli-Q water and stored at −80 • C. Viral replication was detected via RT-qPCR using a Rotor-Gene Q thermal cycler (Qiagen) and the QuantiTect ® Probe PCR Kit (Qiagen). Forward primer 2019-nCoV_N2-F (5 -TTA CAA ACA TTG GCC GCA AA-3 ), reverse primer 2019-nCoV-N2-R (5 -GCG CGA CAT TCC GAA GAA-3 ) and the N2 probe 2019-nCoV_N2-P (FAM-ACA ATT TGC CCC CAG CGC TTC AG-BHQ1) were obtained from Eurofins Genomics (Ebersberg, Germany). The primer and probe sequences were used as recommended by the Centers for Disease Control and Prevention (CDC) in February 2020 [39]. The total volume of each RT-qPCR reaction was 25 µL. The thermal profile was 50 • C for 30 min, followed by 95 • C for 15 min and 45 cycles of 95 • C for 3 s and 55 • C for 30 s.

Data Analysis
Data analysis, copy number calculations, statistics and graphical presentations were performed with Dotmatics GraphPad Prism 9 (Boston, Massachusetts). Statistical differences between groups were determined using Kruskal-Wallis test corrected for multiple comparisons. Symbol meaning are: ns = p > 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. The virus copy numbers were calculated using a calibration curve based on a certified RNA standard (VR-1986D TM from 2019 Novel Coronavirus, Lot: 70035624, ATCC, Glasgow, UK). This commercially available standard, containing 4.73 × 10 3 genome copy numbers per 1 µL, was serially diluted and analyzed by RT-qPCR. The resulting cq-values were plotted against ln[copy numbers], and the equation obtained from a simple linear regression analysis was used to calculate the copy numbers from the cq-values. The standard curve to calculate between cq-values and virus copy numbers is the equation: y = 1.51x + 38.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.